The cytochrome P450s comprise a large gene superfamily that encodes over 500 distinct heme-thiolate proteins that catalyze the oxidation of drugs and numerous other compounds in the body. It is of considerable interest in the pharmaceutical and other fields to identify cytochrome P450s and the role they play in the metabolism of individual compounds. Cytochrome P450s are heme-containing enzymes that strongly absorb at a wavelength of 450 nm when the heme is bound to a molecule of carbon monoxide. They are most well known for their ability to catalyze the metabolism of a wide variety of drugs, xenobiotics, carcinogens, mutagens and pesticides, and they are also involved in catalyzing reactions that make or degrade cholesterol, steroids, and other lipids. The reactions performed by these enzymes are generally oxidations, hydroxylations, acetylations, and demethylations. Mutations in cytochrome P450s or abnormal expression levels can cause a number of human diseases such as glaucoma and breast cancer. Cytochrome P450s are also involved in the metabolism of a number of vitamins, such as Vitamin A (retinoic acid) [White et. al. (1996) J. Biol. Chem. Nov. 22: 271(47): 29922-7; WO197/49815; WO 01/44443] and Vitamin D [Jones, G. et. al. (1999) July; 140(7):3303-10; Dilworth F J, et. al. (1995) July 14; 270(28); 16766-74. In particular, cytochrome P450s, CYP27A, CYP27B and CYP24, are involved in Vitamin D3 metabolism. Vitamin D3, a seco-steroid, is metabolized into its active form by CYP27A and CYP27B and is then further metabolized by CYP24. CYP24 is a mitochrondrial cytochrome P450 that has previously been characterized. For example, isolated human CYP24 was published in Chen et al. (Isolation and expression of human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA. Proc Natl Acad Sci USA 1993 May 15;90(10):4543-7). In Chen et al. it was reported that the human 24-hydroxylase 1539 base pair open reading frame encoded a 513 amino acid sequence, 90% homologous to rat Cyp24. Mouse Cyp24 was characterized in Yoshimura et al. (Molecular cloning of 25-hydroxyvitamin D-3 24-hydroxylase (Cyp-24) from mouse kidney: its induciblity by vitamin D-3. Biochim Biophys Acta 1995 Oct. 17; 1264(1):26-8).
The vitamin D metabolic pathway is part of a vital endocrine system that is highly regulated at certain stages and produces metabolites that control the secretion of the parathyroid gland hormones. 1α,25(OH)2D3, a hormone produced in the vitamin D pathway, regulates phosphate and calcium levels in the blood which in turn control bone mass, the state of bones, and affect cellular differentiation in the skin and the immune system. In the vitamin D pathway, cytochrome P450s introduce functional groups by hydroxylation usually at positions 1, 25, and 24 of the steroid.
The metabolism of vitamin D begins with 25-hydroxlyation of vitamin D3 or D2 in the liver to 25(OH)D3. 25(OH)D3 and a second metabolite, 1α,25(OH)2D3, are converted to 24,25(OH)2D3 and 1,24,25(OH)3D3 by CYP24, a mitochondrial P450 involved in the vitamin D pathway, respectively. CYP24 expression is induced by 1,25(OH)2D3 and is found in the kidney as well as other vitamin D target tissues such as the parathyroid cells, keratinocytes, osteoblasts, and enteroctyes.
There are a number of vitamin D related medical conditions. More information on vitamin D conditions can be found in the Proceedings of the Workshop on Vitamin D (Walter de Gruyter publishing, Berlin), proceedings 1 to 11. For instance, vitamin D deficiency has been related to the following:                1. in the parathyroid—hyper- and hypo-parathyroidism, pseudohypo-parathyroidism, secondary hyperparathyroidism;        2. in the pancreas—diabetes;        3. in the thyroid—medullary carcinoma;        4. in the skin—psoriasis;        5. in the lung—sarcoidosis and tuberculosis;        6. in the kidney—chronic renal disease, glomerulonephritis, IgA nephropathy, membraneous nephropathy, glomerulosclerosis, nephrosis, renal insufficiency, hypophosphtatemic VDRR, vitamin D dependent rickets;        7. in the bone—anticonvulsant treatment, fibrogenisis imperfecta ossium, osteitits fibrosa cystica, osteomalacia, hypocalcemia, osteoporosis, osteopenia, osteosclerosis, renal osteodytrophy, rickets;        8. in the intestine—glucocorticoid antagonism, idopathic hypercalcemia, malabsorption syndrome, steatorrhea, tropical sprue;        9. in the prostate—cancer; and        10. in the breast—cancer.        
More common conditions related to vitamin D or vitamin D metabolite deficiency are obesity problems, hyperphosphatemic tumoral calcinosis, sarcoidosis, tuberculosis, primary hyperparathyroidism, vitamin D dependent rickets type 11, cholestatic or paremchymal liver disease.
Since CYP24 is involved in maintaining vitamin D homeostasis and is implicated in the development of these diseases, it is important to understand how CYP24 activity is and can be modulated in vivo and in vitro. There is also a need for drug design and drug screening methods to identify substances that modulate CYP24.